This invention relates to recombinant DNA technology. In particular, this invention concerns the cloning of nucleic acids encoding multiple drug resistance proteins of Drosophila melanogaster and Anopheles gambiae. More particularly, this invention provides isolated nucleic acid compounds encoding multiple drug resistance proteins of Anopheles gambiae and Drosophila melanogaster. Vectors and transformed host cells comprising the multiple drug resistance-encoding DNA of Anopheles gambiae and Drosophila melanogaster are also provided. The invention further provides assays, which utilize these transformed host cells.
The multidrug-resistance associated protein, MRP1, a large (190 kDa) membrane glycoprotein, was identified in 1992 in a human small-cell lung cancer cell line where its overexpression conferred resistance to a large spectrum of drugs (Cole et al., 1992). MRP joined the P-glycoprotein MRP1, the original protein associated with broad resistance, discovered in 1976 (Juliano & Ling, 1976). The description of MRP1 was followed by the characterization of several other structurally related human proteins: MRPs 2 to 8; followed by MRP9 (Bera et al., 2001; Büchler et al., 1996; Dean et al., 2001; Hopper et al., 2001). These proteins, like MRP1, are members of the ATP-binding cassette (ABC) superfamily, present from bacteria to man, and involved in the energy-dependent transmembrane transport of a variety of molecules, ranging from inorganic ions to large polypeptides. Like other ABC-transporters, MRP homologs are well conserved in evolution and have been described in several mammals (Büchler et al., 1996; Kool et al., 1997; van Aubel et al., 1998), in the nematode Caenorhabditis elegans (Broeks et al., 1996), in the protozoan parasite Leishmania (Essodaigui et al., 1999), in yeast (Szczypka et al., 1994), and in plants (Lu et al., 1997).
ABC transporters are typically composed of two membrane spanning domains (MSDs), containing several transmembrane α-helices, and two cytosolic nucleotide binding domains (NBDs), responsible for the hydrolysis of ATP, thus providing the necessary energy for substrate transport. MRP1 is one of several members of the MRP subfamily characterized by a third MSD of unknown function at the N-terminus of the protein (Borst et al., 2000). The NBDs are highly conserved and share two sequence motifs, designated “Walker A” and “Walker B”, with other nucleotide binding proteins. These sequences are separated by a stretch of about 120–170 amino acids, including a short (12–13 amino acid) peptide motif called the ABC transporter “signature” region. In contrast, the MSDs are highly divergent and are probably involved with the protein's substrate specificity.
Notwithstanding these advances in the art, there continues to be a need in the art to identify MRPs in other species. For example, the identification of MRPs in insects could aid in the development of more effective insecticides.